Metal complexes with a charged linker

ABSTRACT

The present invention concerns new metal complexes with a charged linker and their use as luminescent marker groups in an immunoassay.

This application is a 371 of PCT/EP95/02920 filed Jul. 24, 1995.

DESCRIPTION

The present invention concerns new metal complexes with a charged linkerand their use as luminescent marker groups in immunoassays.

Luminescent metal complexes are known from the state of the art. EP-A-0178 450 discloses ruthenium complexes that are coupled to animmunologically active material in which the ruthenium complexes containthree identical or different bicyclic or polycyclic ligands with atleast two nitrogen-containing heterocycles and at least one of theseligands is substituted with at least one water-solubilizing group suchas --SO₃ H or --COOH and at least one of these ligands is substituteddirectly or via a spacer group with at least one reactive group and theligands are bound via nitrogen atoms to the ruthenium.

The groups capable of coupling are introduced in a very complicatedmanner by activation and consecutive reactions on the solubilizinggroups of the ligands. The production of monoactivated compounds whichenable coupling to biological substances such as antibodies withoutcross-linking also proves to be particularly complicated.

EP-A-0 580 979 discloses the use of osmium and ruthenium complexes asmarker groups for electrochemiluminescence. Heterocycles containingnitrogen such as bipyridines are mentioned as ligands for thesecomplexes. WO 87/06706 discloses further metal complexes which aresuitable as marker groups for electrochemiluminescence measurements.

Further disadvantages of the known metal complexes of the state of theart are a poor quantum yield in electrochemiluminescence measurementsdue to oxygen quenching and photodissociation or/and a high unspecificbinding to proteins.

Therefore the object underlying the present invention was to at leastpartially eliminate the disadvantages of the state of the art.

Surprisingly it was found that the introduction of free positive or/andnegative charge carriers into the linker which links the reactivecoupling group of the metal complex to one of the ligands reduces theadsorption of conjugates of these complexes with an immunologicallyreactive substance and thus also improves the stability and recovery ofthe conjugates in immunoassays. Moreover an increased quantum yield canbe achieved.

Furthermore it was found that luminescent metal complexes with a chargedlinker can be produced in a surprisingly simple manner.

One subject matter of the present invention is thus a metal complex ofthe general formula (I):

     M(L.sub.1 L.sub.2 L.sub.3)!.sub.n -X.sub.m A              (I)

in which M is a divalent or trivalent metal cation selected from rareearth or transition metal ions, L₁, L₂ and L₃ are the same or differentand denote ligands with at least two nitrogen-containing heterocycleswherein L₁, L₂ and L₃ are bound to the metal cation via nitrogen atoms,X is a reactive functional group which is covalently bound to at leastone of the ligands L₁, L₂ and L₃, n is an integer from 1 to 10, m is aninteger from 1 to 6 and is preferably 1 to 3 and A denotes thecounterions which may be required to balance the charge wherein thelinker contains at least one positive or/and negative charge carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 show preferred compounds of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The metal complex is preferably a luminescent metal complex i.e. a metalcomplex which can generate a detectable luminescence reaction. Thisluminescence reaction can for example be detected by fluorescence or byelectrochemiluminescence measurement. The metal cation in this complexis for example a transition metal or a rare earth metal. The metal ispreferably ruthenium, osmium, rhenium, iridium, rhodium, platinum,indium, palladium, molybdenum, technetium, copper, chromium or tungsten.Ruthenium, iridium, rhenium, chromium and osmium are particularlypreferred. Ruthenium is most preferred.

The ligands L₁, L₂ and L₃ are ligands containing at least twonitrogen-containing heterocycles. Aromatic heterocycles are preferredsuch as bipyridiyl, bipyrazyl, terpyridyl and phenanthrolyl. The ligandsL₁, L₂ and L₃ are preferably selected from bipyridine and phenanthrolinering systems.

The reactive functional group X of the complex is a reactive group thatcan be coupled to an immunological active substance. The group X ispreferably an activated carboxylic acid group such as a carboxylic acidhalogenide, a carboxylic acid anhydride or an active ester e.g. anN-hydroxysuccinimide ester, p-nitrophenyl ester, pentafluorophenylester, imidazolyl ester or N-hydroxybenzotriazolyl ester a maleimide ora group which can be photoactivated e.g. an azide. The complexpreferably contains only a single functional group X.

In addition the complex can optionally contain one or severalcounterions A to balance the charge. Examples of suitable negativelycharged counterions are halogenides, OH⁻, carbonate, alkyl carboxylatee.g. trifluoroacetate, sulfate, hexafluorophosphate andtetrafluoroborate groups. Hexafluorophosphate, trifluoroacetate andtetrafluoroborate groups are particularly preferred. Examples ofsuitable positively charged counterions are monovalent cations such asalkali metal and ammonium ions.

The metal complex according to the invention differs from the metalcomplexes known from the state of the art in that it contains at leastone charge carrier in the linker between the ligand and the reactivegroup X capable of coupling. The term "charge carrier" denotes withinthe sense of the present invention a group which is present mainly in anionic form at a pH value in the range 6-8. The linker preferablycontains up to 10, particularly preferably 2-8 and most preferably 2-4such charge carriers.

The linker particularly preferably contains at least one negative chargecarrier. Examples of suitable negative charge carriers are phosphate,phosphonate, sulfonate and carboxylate groups, of which carboxylategroups are most preferred.

Examples of positive charge carriers are amino groups and singly ormultiply substituted amino groups such as mono, di or trialkyl aminogroups in which alkyl denotes a straight-chained or branched alkylresidue of 1-6 C atoms or a cyclic alkyl residue of 3-6 C atoms. Thepositive charge carriers are particularly preferably selected from basicamino acids such as lysine or substituted amino acids such asdiethyllysine. Amines and substituted amines can also serve as electrondonors in the detection of the metal complexes byelectrochemiluminescence.

The linker preferably has a chain length of 4-40 atoms and is analkylene chain modified by the incorporation of heteroatoms such asamide functions. The charge carriers are preferably arranged in thelinker in such a way that an H atom of an alkylene unit is replaced by acharged group e.g. NH₂ ⁺ or CO₂ ⁻.

The linker which contains the free charge carriers is preferablycomposed at least partially of amino-carboxylic acid units which arelinked together via peptide bonds. In such a linker the charge carrierscan be derived from free amino or/and carboxylate groups ofpolyfunctional aminocarboxylic acids which contain a total of at leastthree charged groups (amino plus carboxylate) so that afterincorporation into the linker and the concomitant reaction of two of thecharged groups at least one free charge carrier is still present. Forexample the charge carriers can be derived from trifunctionalaminocarboxylic acids which contain (a) an amino group and twocarboxylate groups or (b) two amino groups and one carboxylate group.Examples of such trifunctional aminocarboxylic acids are lysine,ornithine, hydrolxylysine, aspartic acid and glutamic acid.

The metal complex according to the invention can additionally contain atleast one hydrophilic group selected from C₂ -C₃ alkylenoxy units, C₂-C₃ alkylenethio units, C₂ -C₃ alkylenamino units and polyhydroxy units.

The polyhydroxy units are preferably selected from groups of formulae(IIa) or (IIb): ##STR1## in which W denotes an organic residue with atleast two hydroxy groups and R denotes hydrogen or C₁ -C₅ alkyl. Theorganic residue W preferably contains 2 to 6 and particularly preferably2 to 4 hydroxy groups. Furthermore W should advantageously contain 2 to10 and in particular 3-6 carbon atoms. Specific examples of suitablepolyhydroxy units are residues of polyalcohols such as glycerol oraminopolyalcohols. A preferred aminopolyalcohol is Tris(2-amino-2-(hydroxymethyl)-1,3-propanetriol). In this case thepolyhydroxy unit has the formula NH--C(CH₂ OH)₃. The polyalcohols oraminopolyalcohols are preferably coupled to the metal complex in theform of esters or amides. The OH groups of the polyalcohol oraminopolyalcohols can be optionally substituted by hydrophilic groupse.g. by dendrimeric groups.

The C₂ -C₃ alkylenoxy, C₂ -C₃ -alkylenethio and C₂ -C₃ -alkylenaminounits of the metal complex according to the invention are preferably C₂units and in particular ethylenoxy units. The complex preferablycontains 1 to 30 and particularly preferably 2 to 20 C₂ -C₃ alkylenoxy,C₂ -C₃ alkylenethio or C₂ -C₃ alkylenamino units per metal cation. Theseunits are components of substituents of the heterocyclic ligands of themetal complex. They can be present in the linker between one of theligands and the reactive functional group X or/and in monosubstituents.The alkylenoxy, alkylenethio or alkylenamino units can also be linkedtogether via a bridgehead which can optionally carry a functional groupX. On the other hand several complex units can also be linked togethervia the bridgehead.

In one embodiment of the present invention the metal complex accordingto the invention has the general formula (III): ##STR2## in which M, Xand A are defined as above, R₁, R₂, R₃, R₄, R₅ and R₆ are the same ordifferent and each denotes one or several substituents provided that Xis linked to one of the ligands via one of the substituents R₁, R₂, R₃,R₄, R₅ and R₆ as the linker.

The ligands of the complex may also be substituted phenanthroline orbipyridine systems depending on the presence or absence of the groupsindicated by the broken lines.

The substituents R₁, R₂, R₃, R₄, R₅ and R₆ on the ligand are--providedthey do not contain the group X of the linker--preferably hydrogen, C₁-C₅ alkyl and in particular C₁ -C₃ alkyl or a hydrophilic group asdefined above.

In a particularly preferred embodiment the metal complexes have thegeneral formula (IIIa): ##STR3## in which M, X and A are defined asabove, R₁, R₂, R₃, R₄ and R₅ are defined as above, s is an integer from0 to 6 and preferably from 1 to 4 and Y denotes the linker with freecharge carriers.

Examples of compounds of formulae (III) and (IIIa) are shown in FIGS.1-3. FIGS. 1 and 2 show complexes which have a linker with one freenegative charge carrier in each case. In each case the linker contains atrifunctional amino acid, namely lysine, whose amino groups serve toform peptide bonds in the linker whereas the carboxylate group forms thefree charge carrier. FIG. 3 shows a linker composed of 4 amino acidunits namely β-alanine, lysine and two glutamic acid residues.

The linker contains three negative charge carriers, one carboxylategroup from each of the two Glu residues and one carboxylate group whichis derived from Lys. The functional group X in FIGS. 1 and 3 is amaleimide suitable for coupling to SH groups and in FIG. 2 it is anN-hydroxysuccinimide ester suitable for coupling to NH₂ groups.

The ligands of the metal complex according to the invention can also belinked together so that the metal complex is present in the form of asemicage or cage. A preferred embodiment of a metal complex according tothe invention in the form of a semicage or cage has the general formula(IV): ##STR4## in which M, X, n and A are defined as above, R₁, R₂ andR₃ are the same or different and each denote one or severalsubstituents--as defined above--on the bipyridine or phenanthrolineligand and Y in each case denotes a linker with at least one chargecarrier.

If the substituents R₁, R₂ and R₃ in formula (IV) are covalently linkedtogether optionally via linker groups, then the complex of formula (IV)is in the form of a cage.

The complex of formula (IV) may not only be present as a monomer but asan oligomer composed of preferably up to 5 individual metal complexes.In this case the functional group X capable of coupling can for examplebe a substituent on an aromatic nucleus. e.g. a phenyl nucleus in whichcase two or several of the remaining substituent positions of thearomatic nucleus can be substituted by a metal complex in the form of asemicage or cage.

The metal complexes according to the invention can be produced byreacting a metal salt e.g. a metal halogenide with the appropriateligands and optionally subsequently replacing the halogenide ion byhexafluorophosphate, trifluoroacetate or tetrafluoroborate anions. Suchprocesses are described in the state of the art e.g. in U.S. Pat. No.4,745,076, U.S. Pat. No. 5,075,447 and U.S. Pat. No. 5,585,279.Reference is hereby made to this disclosure.

The synthesis of a charged linker on an N-heterocyclic ligand with acharged linker can on the one hand be achieved as a coupling reaction insolution in which an optionally partially protected aminocarboxylic acidis coupled to a reactive group of the ligand e.g. a carboxylic acid.This coupling section can if necessary be repeated again until a linkerof the desired length has been synthesized. In this process at least onepolyfunctional aminocarboxylic acid group is introduced which containsone or several charged side groups.

Subsequently the reactive group X is introduced and protecting groupswhich may be present on the side groups of the aminocarboxylic acids arecleaved off. This synthesis of the ligand by successive coupling ofamino acids in solution can on the one hand be carried out on a singleligand and, on the other hand, on a ligand as starting material which isalready bound to a metal complex. A suitable starting material is forexample a luminescent metal complex which contains a free carboxylategroup. Such complexes are known from the above-mentioned documents andare commercially available for example from Igen Inc. Co., Rockville,Md., USA.

On the other hand the complexes can also be synthesized by solid phasepeptide synthesis. In a first embodiment of the solid phase synthesis anamino acid is coupled to the solid phase support via its carboxylategroup and the desired linker is then synthesized by successive couplingof further amino acids. In order to produce a linker according to theinvention at least one amino acid is used in this process which containsa charged group as a side group e.g. an amino or carboxylate group,optionally in a protected form. After the desired linker sequence hasbeen completed, an activated metal complex e.g. in the form of an activeester, can be coupled to the free N-terminal amino group of the solidphase-bound peptide. After cleavage from the solid phase the reactivegroup X can be coupled to the carboxy terminus of the peptide linker andprotecting groups which may be present are cleaved.

In another embodiment of the solid phase synthesis an amino acid-metalcomplex conjugate which contains a protected amino group and acarboxylate group e.g. Fmoc-Lys (BRu)--OH (FIG. 4), can be anchored to asolid phase via the free carboxylate group and a peptide linker can besynthesized after releasing the blocked amino group. After finishing thedesired linker sequence the complex is cleaved from the solid phase toobtain a linker which contains at least the original carboxylate anchorgroup as the free charge carrier. The reactive group X can be coupled tothe amino terminus of the resulting peptide linker.

In a third embodiment of the solid phase synthesis the linker sequencewith charge carriers can also be synthesized directly on a selectedpeptide epitope.

A combination of the above-mentioned synthesis variants can also be usedto produce the metal complexes according to the invention. Aminoacid-metal complex conjugates that are suitable for the solid phasesynthesis of the complexes according to the invention with a chargedlinker are described in DE-A-44 30 998.8. Reference is hereby made tothis disclosure.

The production of metal complexes of formula (IV) with a semicage orcage structure can for example be carried out by attaching chargedlinkers to the bipyridine or phenanthroline ligands and linking theseunits to a bridgehead via an amide bond. If two bridgeheads are used itis possible to obtain complete cage structures. The linking of threeligands to a trivalent bridgehead e.g. Tris is preferred.

A further subject matter of the present invention is a conjugatecomprising a biological substance to which at least one metal complexaccording to the invention is coupled. Examples of suitable biologicalsubstances are cells, viruses, subcellular particles, proteins,lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids,peptidic nucleic acids (PNA), oligosaccharides, polysaccharides,lipopolysaccharides, cellular metabolites, haptens, hormones,pharmacologically active substances, alkaloids, steroids, vitamins,amino acids and sugars.

The metal complex is coupled to the biological substance via thereactive functional group of the metal complex which can covalentlycouple to a functional group of the biological substance. If thefunctional group is an active ester it can for example be coupled to thefree amino groups of the biological substance. If the functional groupis a maleinimide residue it can be coupled to the free SH groups of thebiological substance.

In a particularly preferred embodiment of the present invention themetal complexes are coupled to a peptide which preferably has a maximumlength of 50 amino acids and particularly preferably of 30 amino acids.The production of these peptides labelled with a metal complex ispreferably carried out by synthesizing a peptide with the desired aminoacid sequence on a solid phase in which a) after the synthesis anactivated metal complex, preferably a metal complex-active esterderivative, is coupled to the N-terminal amino group of the peptideor/and b) during the synthesis an amino acid derivative that iscovalently coupled to a metal complex is introduced in at least oneposition of the peptide. The coupling of the metal complex to theN-terminal amino acid of the peptide is preferably carried out beforecleaving the peptide from the solid phase and before cleaving protectinggroups on reactive side groups of the amino acid derivatives used forthe peptide synthesis.

The peptides preferably contain an immunologically reactive epitoperegion and a spacer region wherein at least one metal complex markergroup is coupled to the spacer region. The spacer region preferably hasa length of 1 to 10 amino acids and is located at the amino or/andcarboxy terminus of the peptide.

The spacer region preferably contains amino acids which have chargesor/and can form hydrogen bridges. The amino acids of the spacer regionare preferably formed from the group comprising glycine, β-alanine,γ-aminobutyric acid, ε-aminocaproic acid, lysine and compounds of thestructural formula NH₂ -- (CH₂)_(y) O!_(x) --CH₂ --CH₂ --COOH in which yis 2 or 3 and x is 1 to 10.

The epitope regions of the peptides are preferably derived frompathogenic organisms e.g. bacteria, viruses and protozoa or fromautoimmune antigens. The epitope region is particularly preferablyderived from viral antigens e.g. the amino acid sequences of HIVI, HIVIIor hepatitis C virus (HCV).

Further preferred examples of biological substances are biotin, nucleicacids, antibodies or antibody fragments, polypeptide antigens i.e.immunologically reactive polypeptides or haptens i.e. organic moleculeswith a molecular weight of 150 to 2000, in particular molecules with asteroid backbone such as cardenolides, cardenolide-glycosides (e.g.digoxin, digoxigenin), steroid alkaloids, sexual hormones (e.g.progesterone), glucocorticoids etc. Further examples of haptens areprostaglandins, leucotrienes, leuco-en-diines, thromboxanes etc.

Yet a further subject matter of the present invention is the use of themetal complexes according to the invention or of the conjugatesaccording to the invention in an immunological detection method or anucleic acid hybridization method, in particular in a luminescenceassay.

In these methods the metal complex is used as a marker group with theaid of which it is possible to qualitatively or/and quantitativelydetermine an analyte in a sample solution. The metal complex ispreferably detected by electrochemiluminescence in which caseluminescent species are generated electrochemically on the surface of anelectrode. Examples for carrying out luminescence assays using metalcomplexes from the state of the art may be found in EP-A-0 580 979, WO90/05301, WO 90/11511 and WO 92/14138. Reference is hereby made to themethods and devices for luminescence assays disclosed therein.Electrochemiluminescence assays are carried out in the presence of asolid phase which is preferably composed of microparticles and inparticular of magnetic microparticles which are provided with a reactivecoating e.g. with streptavidin. In this manner immune or hybridizationcomplexes containing a metal complex as a marker group can be detectedbound to a solid phase.

The electrochemiluminescence measurement is preferably carried out inthe presence of a reducing agent for the metal complex e.g. an amine.Aliphatic amines and in particular primary, secondary and tertiaryalkylamines, the alkyl groups of which each have 1 to 3 carbon atoms arepreferred. Tripropylamine is particularly preferred. The amine can,however, also be an aromatic amine such as aniline or a heterocyclicamine. The reducing agent can already be integrated into the ligandsphere of the complex.

In addition a non-ionic surface-active agent may also be present as anamplifier e.g. an ethoxylated phenol. Such substances are for examplecommercially available under the names Triton X100 or Triton N401.

On the other hand the luminescent metal complex can also be detected byfluorescence in which case the metal chelate is excited by irradiationwith light of a suitable wavelength and the resulting fluorescentradiation is measured. Examples for carrying out fluorescence assays maybe found in EP-A-0 178 450 and EP-A-0 255 534. Reference is hereby madeto this disclosure.

The previously described principle of using metal complexes with chargedlinkers can in addition also be applied to other marker or/and solidphase binding groups. The present invention therefore also concernsmarker or/and solid phase binding groups with a reactive functionalgroup X covalently bound via a linker wherein the linker contains atleast one positive or/and negative charge carrier. The linker ispreferably composed at least partially of aminocarboxylic acid unitswhich are linked together via peptide bonds.

The marker or/and solid phase binding groups are preferably selectedfrom fluorescent marker groups such as e.g. fluorescein, coumarin,rhodamine, resorufin, cyanine and derivatives thereof as well as biotinand biotin analogues such as iminobiotin or desthiobiotin.

The invention also concerns conjugates of the aforementioned markeror/and solid phase binding groups with a biological substance as definedpreviously. The marker or/and solid phase binding groups or conjugatesthereof can be used in an immunological detection method or in a nucleicacid hybridization method. In these methods it is possible to achieve animprovement in the solubility and a reduction or prevention of signalquenching due to the formation of dimers or higher aggregates.

Yet a further subject matter of the present invention is a process forintroducing an N-hydroxysuccinimide ester group into peptides or peptidederivatives which is characterized in that a peptide or peptidederivative which has a free amino function, preferably an aliphatic andparticularly preferably a primary aliphatic amino function, is reactedwith suberic acid-bis-hydroxysuccinimide ester (DSS) thus converting theamino function into an N-hydroxysuccinimide ester. The peptide orpeptide derivative activated by introducing the N-hydroxysuccinimideester group can subsequently be coupled to biological substances asdefined above.

A particular advantage of this process is that peptides or peptidederivatives can be used which have at least one free acid function andin particular a carboxylic acid function. In contrast to known processesit is not necessary to block the acid function.

In addition to peptides one can also activate those peptide derivativeswhich carry at least one marker or/and solid phase binding group e.g. ametal complex, a fluorescent group or a biotin group.

The activation reaction is preferably carried out in an organic solventsuch as dimethylformamide in the presence of a base e.g. a tertiaryamine such as triethylamine. The suberic acid-bis-N-hydroxysuccinimideester is preferably used in a molar excess of 2:1 to 10:1 relative tothe peptide or peptide derivative.

The present invention is further elucidated by the following examplesand figures.

FIGS. 1-3 show metal complexes according to the invention

FIG. 4 shows an amino acid metal complex conjugate suitable forsynthesizing a metal complex according to the invention and

FIG. 5 shows an activated peptide-metal complex conjugate.

EXAMPLE 1

Production of a metal complex with a charged linker

6 mmol of the metal complex Ru(bipyridine)₂-(bipyridine-CO-N-hydroxysuccinimide ester) according to EP-A-0 580 979is dissolved in 50 ml dimethylformamide and a solution of Fmoc-lysine indimethylformamide is added dropwise. The solvent is removed in a highvacuum. The residue is dissolved in a small amount of acetone, admixedwith 300 ml chloroform and briefly heated to boiling point. It isallowed to cool and the separated oil is separated from the solvent. Thedesired compound Ru(bpy)₂ (bpy-CO-(Fmoc)-Lys) is obtained as a solidsubstance by drying.

The Fmoc protecting group is cleaved by reaction for 2 hours at 80° C.in dioxane/acetone in a 4-fold excess of piperidine. After cooling theoily residue is separated and a chloroform/water extraction is carriedout. The aqueous phase is isolated and the compound Ru(bpy)₂(bpy-CO-Lys) is obtained as a red solid.

60 mg of this compound is dissolved in 10 ml acetone, maleimidopropionicacid-N-hydroxysuccinimide ester is added and it is stirred for 4 hoursat room temperature. The residue is purified by preparative HPLC. 17 mgof the compound Ru(bpy)₂ (bpy-CO-Lys-MP) is obtained. This compound isshown in FIG. 1.

EXAMPLE 2

Production of a metal complex with a charged linker

Ru(bpy)₂ (bpy-CO-Lys) according to example 1 and a 10-fold molar excessof suberic-bis-N-hydroxysuccinimide ester are dissolved indimethylformamide and stirred for 2 hours at room temperature. Thesolvent is removed in a high vacuum, the residue is extracted with waterand lyophilized after treatment with hexane. It is purified by means ofHPLC (100% H₂ O, in 20 min to 100% acetonitrile, C₁₈, 3 μm column, flowrate: 1 ml/min, retention time: 10.00 min.). The compound obtained isshown in FIG. 2.

EXAMPLE 3

Production of metal complexes by means of solid phase peptide synthesis

The metal complexes with a charged linker were produced by means offluorenylmethyloxycarbonyl-(Fmoc)-solid phase peptide synthesis on abatch peptide synthesizer e.g. from Applied Biosystems A431 or A433. Forthis 4.0 equivalents of the amino acid derivatives shown in Table 1 wereused in each case.

                  TABLE 1    ______________________________________    A             Fmoc-Ala-OH    C             Fmoc-Cys(Trt)-OH    D             Fmoc-Asp(OtBu)-OH    E             Fmoc-Glu(OtBu)-OH    F             Fmoc-Phe-OH    G             Fmoc-Gly-OH    H             Fmoc-His(Trt)-OH    I             Fmoc-Ile-OH    K1            Fmoc-Lys(Boc)-OH    K2            Boc-Lys(Fmoc)-OH    K3            Fmoc-Lys(BPRu)-OH    L             Fmoc-Leu-OH    M             Fmoc-Met-OH    N             Fmoc-Asn(Trt)-OH    P             Fmoc-Pro-OH    Q             Fmoc-Gln(Trt)-OH    R             Fmoc-Arg(Pmc)-OH    S             Fmoc-Ser(tBu)-OH    T             Fmoc-Thr(tBu)-OH    U             Fmoc-Balanine-OH    V             Fmoc-Val-OH    W             Fmoc-Trp-OH    Y             Fmoc-Tyr(tBu)-OH    Z             Fmoc-ε-aminocaproic acid-OH    Nle           Fmoc-ε-norleucine-OH    Abu           Fmoc-γ-aminobutyric acid-OH    ______________________________________

In the variant (a)--introduction of the metal complex after completionof the solid phase synthesis--an activated ruthenium(bipyridyl)₃ complex(BPRu) e.g. Ru(bpy)₂ (bpy-CO-NHS) (cf. example 1) was coupled to theN-terminal amino acid of the peptide.

According to variant (b) metal chelate groups were introduced into thepeptide sequence by direct incorporation of metal chelate-coupled aminoacid derivatives e.g. at the carboxyl terminus of the sequence via alysine residue ε-derivatized with a metal chelate active ester e.g. thelysine derivative K3 (FIG. 4).

The amino acids or amino acid derivatives were dissolved inN-methylpyrrolidone. The peptide was synthesized on 400-500 mg(4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (TetrahedronLetters 28 (1987), 2107) loaded with 0.4-0.7 mmol/g (JACS 95 (1973),1328). The coupling reactions were carried out for 20 min. with 4equivalents dicyclohexylcarbodiimide and 4 equivalentsN-hydroxybenzotriazole with respect to the Fmoc-amino acid derivative indimethylformamide as the reaction medium. The Fmoc group was cleavedwithin 20 min. after each synthesis step using 20% piperidine indimethylformamide.

When cysteine residues were present in the peptide sequence, the solidphase was oxidized immediately after completing the synthesis usingiodine in hexafluoroisopropanol/dichloromethane.

The peptide was released from the support and the acid-labile protectinggroups were cleaved using 20 ml trifluoroacetic acid, 0.5 mlethanedithiol, 1 ml thioanisole, 1.5 g phenol and 1 ml water in 40 min.at room temperature. The reaction solution was subsequently admixed with300 ml cooled diisopropyl ether and kept at 0° C. for 40 min. untilcomplete precipitation of the peptide. The precipitate was filtered,rewashed with diisopropyl ether, dissolved in a small amount of 50%acetic acid and lyophilized. The crude material obtained was purified bymeans of preparative HPLC on Delta-PAK RP C18 material (column 50×300mm, 100 Å, 15 μ) over a corresponding gradient (eluant A: water, 0.1%trifluoroacetic acid, eluant: B acetonitrile, 0.1% trifluoroacetic acid)in ca. 120 min. The identity of the eluted material was checked by meansof ion spray mass spectrometry.

The metal chelate label was introduced on the free N-terminal aminogroup of the support-bound peptide according to variant (a) viaappropriate active ester derivatives. For this 4 equivalents ruthenium(bipyridyl)₃ complex (BPRu) per free primary amino function wereactivated with N-hydroxybenzotriazole/dicyclohexylcarbodiimide,dissolved in a small amount of DMSO, added dropwise and stirred at roomtemperature. The reaction was monitored by means of analytical HPLC.After cleaving from the support, the product was purified by preparativeHPLC. The identity of the eluted material was checked by means of ionspray mass spectrometry.

The peptides were synthesized by a combination of variant (a) and (b)i.e. incorporation of metal-chelate-coupled amino acid derivativeswithin the sequence, cleaving the N-terminal Fmoc group and reacting thefree N-terminal amino group with a metal-chelate active esterderivative.

When the metal chelate-coupled amino acid derivatives were exclusivelyincorporated directly during the solid phase synthesis according tovariant (b), it was no longer necessary to subsequently introduce metalchelate active esters.

An example of a metal complex produced by solid phase synthesis is shownin FIG. 3.

In order to introduce the maleinimide function the peptide was dissolvedin 0.1 M potassium phosphate buffer pH 7.0 and admixed with oneequivalent of maleinimide propionic acid-N-hydroxysuccinimide ester inDMSO and stirred for 16 hours at 25° C. The mixture was purified bymeans of preparative HPLC (see above). The identity of the elutedmaterial was checked by means of ion spray mass spectrometry. A reactiveN-hydroxysuccinimide ester function was introduced according to DE-A-4302 241.

EXAMPLE 4

Use of metal complexes with a charged linker in immunological tests

A double-antigen bridge test was carried out to detect specificantibodies against hepatitis C virus (HCV). For this the sample liquidwas incubated with a ruthenium-labelled antigen and a biotinylatedantigen against the antibody to be determined in the presence of a solidphase coated with streptavidin. The presence of anti-HCV antibodies inthe sample liquid was determined by determining the label in the solidphase by electrochemiluminescence according to the Flash system.

A HCV polypeptide was used as an antigen which contains the amino acids1207-1488 of HCV. The amino acid sequence and the synthesis of such apolypeptide is described in DE-A-44 28 705.4.

In order to derivatize the HCV polypeptide with ruthenium complexesactivated with succinimide ester, the polypeptide was dissolved in a 100mM sodium phosphate buffer pH 6.5, 0.1% SDS at a protein concentrationof 10 mg/ml. The pH value was set to 8.5 by addition of 5 M NaOH and thesolution was supplemented with dithiothreitol to a final concentrationof 2 mM. The amount of a ruthenium complex activated with a succinimideester in DMSO that corresponds to the desired offered stoichiometry wasadded to this solution and it was subsequently incubated for 60 min at65° C. while stirring. The reaction was terminated by supplementing thereaction mixture with lysine to a final concentration of 10 mM andincubating it for a further 30 min. Subsequently the mixture wasdialysed against 100 mM sodium phosphate buffer pH 6.5, 0.1% SDS. Theresulting protein solution was admixed with sucrose (final concentration6.5% (w/v)) and lyophilized in portions.

For the production of a HCV polypeptide derivatized with a rutheniumcomplex activated with maleinimide, the polypeptide was taken up in 100mM sodium phosphate buffer pH 6.5, 0.1% SDS (protein concentration 10mg/ml). An amount of a maleinimide-activated ruthenium complex in DMSOthat corresponds to the desired offered stoichiometry was added to thissolution and it was incubated for 60 min at 25° C. while stirring. Thereaction was terminated by supplementing the reaction mixture withcysteine to a final concentration of 10 mM and further incubating it for30 min. Afterwards the reaction mixture was dialysed as described above,admixed with sucrose and lyophilized in portions.

Three experiments were carried out in which different ruthenylatedantigens were used each time. For experiment A (comparison) thepolypeptide was coupled to the ruthenium complex according to EP-A-0 580979 used as the starting material in examples 1 and 2 in astoichiometric ratio of 1:3. For experiment B the polypeptide wascoupled to the ruthenium complex according to the invention (FIG. 1)produced in example 1 in a stoichiometric ratio of 1:1. For experiment Cthe polypeptide was coupled to the ruthenium complex (FIG. 3) producedin example 3 in a stoichiometric ratio of 1:1. In all 3 experiments apolypeptide was used as the biotinylated antigen which had been coupledto a maleinimide-activated biotin in a stoichiometric ratio of 1:6. Theruthenylated and biotinylated antigens were in each case used at aconcentration of 400 ng/ml test liquid.

The results of experiments A, B and C are shown in Table 2 in ECLcounts. It can be seen that a reliable differentiation between anegative serum sample and a critical positive serum sample can only beachieved when using the metal complexes according to the invention witha charged linker as marker groups. This is shown by a higherpositive/negative ratio.

                  TABLE 2    ______________________________________    Experiment  A (comparison)                             B        C    ______________________________________    negative sample                323317       132288   14467    positive sample                465769       1323338  319752    Ratio       1.4          10       22    positive/negative    ______________________________________

EXAMPLE 5

Use of metal complexes and biotin groups with a charged linker inimmunological tests

A double-antigen bridge test was carried out to detect specificantibodies against HIV I according to the protocol of example 4.

The HIV polypeptide gp32 was used as the antigen.

The incorporation of the peptide linker sequences "EEE" and "EEEUZU"between the antigen and the ruthenium complex or/and biotin led to asignificant decrease of the unspecific signal compared to antigenswithout a linker sequence whereas the specific signal was essentiallymaintained in HIV-positive samples. Thus significantly improved dynamicsin the determination of analytes can be achieved by using marker or/andsolid phase binding groups with charged linkers.

EXAMPLE 6

Introduction of N-hydroxysuccinimide ester groups into peptidederivatives

250 mg suberic acid-bis-N-hydroxysuccinimide ester (DSS) was dissolvedtogether with 50 μl triethylamine in dimethylformamide. A solution ofthe peptide derivative BPRu-UEEK (100 mg in DMF) which had been preparedaccording to the standard method described in example 3 was addeddropwise to this. After ca. 15 min the DMF was removed in a high vacuum,the residue was taken up in water and the undissolved DSS was removed byfiltration. The filtrate was lyophilized.

The product shown in FIG. 5 was obtained. The purity was 91% accordingto HPLC. Analysis by means of NMR and MS corresponded to the expectedproduct.

A twice ruthenylated peptide derivative having the sequence Ac-K (BPRu)UEUEUK-(DSS)-UEUEUK (BPRu) UE SEQ ID NO. 1! was prepared in an analogousmanner. Biotinylated peptides or peptides provided with other markergroups or unlabelled peptides can be activated with DSS in an analogousway instead of ruthenylated peptides. Surprisingly the reaction proceedssmoothly in the presence of free carboxylic acid functions.

    __________________________________________________________________________    #             SEQUENCE LISTING    - <160> NUMBER OF SEQ ID NOS: 1    - <210> SEQ ID NO 1    <211> LENGTH: 15    <212> TYPE: PRT    <213> ORGANISM: Artificial Sequence    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (1)    #group and aINFORMATION: Modified with an acetyl          ruthenium(bipridyl) complex    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (2)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (4)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (6)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (7)    <223> OTHER INFORMATION: Modified with a acid-bis- - #N-hydroxysuccinimide          ester (DSS)    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (8)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (10)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (12)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (13)    <223> OTHER INFORMATION: Modified with a ruthenium - #(bipridyl) complex    <220> FEATURE:    <221> NAME/KEY: MOD.sub.-- RES    <222> LOCATION: (14)    <223> OTHER INFORMATION: Xaa is Fmoc-B alanine-OH    <220> FEATURE:    #Sequence: PeptideATION: Description of Artificial          Derivative    - <400> SEQUENCE: 1    - Lys Xaa Glu Xaa Glu Xaa Lys Xaa Glu Xaa Gl - #u Xaa Lys Xaa Glu    #                 15    __________________________________________________________________________

We claim:
 1. A metal complex having the formula:

     M(L.sub.1 L.sub.2 L.sub.3)!.sub.n - Y-X!.sub.m

wherein M is a divalent or trivalent metal cation selected from thegroup consisting of a rare earth metal cation and a transition metalcation; L₁, L₂ and L₃ are the same or different, and each is a ligandhaving at least two nitrogen-containing heterocyclic rings, wherein L₁,L₂ and L₃ are bound to the metal cation via nitrogen atoms; Y is alinker, connected to at least one of L₁, L₂ and L₃, the linkercontaining at least one charge carrier independently selected from thegroup consisting of a positive charge carrier and a negative chargecarrier; X is a reactive functional group; n is 1 to 10; and m is 1 to6.
 2. The metal complex as claimed in claim 1, wherein M is selectedfrom the group consisting of ruthenium ion, rhenium ion, osmium ion,chromium ion and iridium ion.
 3. The metal complex as claimed in claim1, wherein M is a ruthenium ion.
 4. The metal complex as claimed inclaim 1, wherein each of L₁, L₂ and L₃ independently contains a ringsystem selected from the group consisting of bipyridine andphenanthroline.
 5. The metal complex as claimed in claim 1, wherein X isselected from the group consisting of carboxylic acid halogenide,carboxylic acid anhydride, active ester, maleimide and a group which iscapable of being photo-activated.
 6. The metal complex as claimed inclaim 1, wherein the metal complex further comprises at least onecounterion A which balances the charge carried by the at least onecharge carrier.
 7. The metal complex as claimed in claim 6, wherein A isat least one group selected from the group consisting ofhexafluorophosphate, trifluoroacetate and tetrafluoroborate.
 8. Themetal complex as claimed in claim 1, wherein Y is a linker containing atleast one negative charge carrier selected from the group consisting ofphosphate, phosphonate, sulfonate and carboxylate.
 9. The metal complexas claimed in claim 1, wherein Y is a linker containing at least onecarboxylate group.
 10. The metal complex as claimed in claim 1, whereinY is a linker containing at least one positive charge carrier selectedfrom the group consisting of amino and substituted amino group.
 11. Themetal complex claimed in claim 10, wherein the at least one positivecharge carrier serves as an electron donor for the metal complex. 12.The metal complex as claimed in claim 1, wherein the linker contains 1to 10 charge carriers.
 13. The metal complex as claimed in claim 1,wherein the linker contains 2 to 8 charge carriers.
 14. The metalcomplex as claimed in claim 1, wherein the linker comprises a pluralityof aminocarboxylic acid units which are linked together via peptidebonds.
 15. The metal complex as claimed in claim 14, wherein theplurality of aminocarboxylic acid units are derived from polyfunctionalaminocarboxylic acids which contain at least one free charge carrierafter incorporation into the linker.
 16. The metal complex as claimed inclaim 14, wherein the plurality of aminocarboxylic acid units arederived from trifunctional aminocarboxylic acids which contain(a) oneamino group and two carboxylate groups, or (b) two amino groups and onecarboxylate group.
 17. The metal complex as claimed in claim 16, whereinthe trifunctional aminocarboxylic acids are selected from the groupconsisting of lysine, ornithine, hydroxylysine, aspartic acid andglutamic acid.
 18. The metal complex as claimed in claim 1, wherein themetal complex has the formula: ##STR5## wherein M, Y and X are as statedabove; and R₁ through R₆, insofar as they are not bound to --Y--X, areeach independently selected from the group consisting of hydrogen, C₁-C₅ alkyl and at least one hydrophilic substituent which is selectedfrom the group consisting of C₂ -C₃ alkyleneoxy, C₂ -C₃ alkylenethio, C₂-C₃ alkyleneamino, a group of formula --NR--W and a group of formula--O--W, wherein W is an organic residue containing 2 to 10 carbon atomsand 2 to 6 hydroxy groups, and R is hydrogen or C₁ -C₅ alkyl,wherein atleast one of R₁ through R₆ is bound to --Y--X, and is represented by theformula --(CH₂)_(s) --C(═O)--NH--(CH₂)₂ --Y--X, wherein s is 0 to
 6. 19.The metal complex as claimed in claim 1, wherein the metal complex hasthe formula: ##STR6## wherein M, Y, X and n are as defined above; and R₁through R₃ are each independently selected from the group consisting ofhydrogen, C₁ -C6 alkyl and at least one hydrophilic group which isselected from the group consisting of C₂ -C₃ alkyleneoxy, C2-C₃alkylenethio, C₂ -C₃ alkyleneamino, a group of formula --NR--W and agroup of formula --O--W, wherein W is an organic residue containing 2 to10 carbon atoms and 2 to 6 hydroxy groups, and R is hydrogen or C₁ -C₅alkyl.
 20. A conjugate, comprising the metal complex as claimed in claim1 coupled with a biological substance.
 21. The conjugate as claimed inclaim 20, wherein the biological substance is selected from the groupconsisting of biotin, an antibody, an antibody fragment, a nucleic acid,a polypeptide antigen, an immunological reactive peptide and a hapten.22. In an immunological detection method wherein a metal complex isbound to an immunological binding partner and used as a marker group toquantitatively or qualitatively determine an analyte in a samplesolution, the improvement comprising using the marker group of claim 1as the marker group.
 23. The method of claim 22, wherein the markergroup is determined by luminescence.
 24. The method of claim 22, whereinthe marker group is determined by electrochemiluminescence.
 25. In animmunological detection method wherein a metal complex is bound to animmunological binding partner and used as a marker group toquantitatively or qualitatively determine an analyte in a samplesolution, the improvement comprising using the conjugate of claim 20 asthe marker group.
 26. The method of claim 25, wherein the marker groupis determined by luminescence.
 27. The method of claim 25, wherein themarker group is determined by electrochemiluminescence.
 28. In a nucleicacid hybridization detection method wherein a metal complex is bound toa probe and used as a marker group to quantitatively or qualitativelydetermine a nucleic acid in a sample solution, the improvementcomprising using the marker group of claim 1 as the marker group. 29.The method of claim 28, wherein the marker group is determined byluminescence.
 30. The method of claim 28, wherein the marker group isdetermined by electrochemiluminescence.
 31. In a nucleic acidhybridization detection method wherein a metal complex is bound to aprobe and used as a marker group to quantitatively or qualitativelydetermine a nucleic acid in a sample solution, the improvementcomprising using the conjugate of claim 20 as the marker group.
 32. Themethod of claim 31, wherein the marker group is determined byluminescence.
 33. The method of claim 31, wherein the marker group isdetermined by electrochemiluminescence.
 34. The metal complex as claimedin claim 18, wherein the metal complex further comprises at least onecounterion A which balances the charge carried by the at least onecharge carrier.
 35. The metal complex as claimed in claim 19, whereinthe metal complex further comprises at least one counterion A whichbalances the charge carried by the at least one charge carrier.